US6403320B1ExpiredUtility

Support bound probes and methods of analysis using the same

Assignee: AFFYMETRIX INCPriority: Jun 7, 1989Filed: Oct 5, 2000Granted: Jun 11, 2002

Est. expiryJun 7, 2009(expired)· nominal 20-yr term from priority

Inventors:J. Leighton Read Stephen P. A. Fodor Lubert Stryer Michael C. Pirrung

B01J 2219/0061B01J 2219/00725B01J 2219/00468B82Y 30/00B01J 2219/00596B01J 2219/00605C07H 19/10C07K 1/045B01J 2219/00315C12Q 1/6816C40B 60/14B01J 2219/00475C07C 229/16B01J 2219/00617B01J 2219/00711B82Y 10/00B01J 2219/005C12Q 1/6837B01J 2219/00621C07D 263/44B01J 2219/00695C07B 2200/11C12Q 1/6809B01J 2219/00626C07C 229/14B01J 2219/00459C07K 7/06B01J 2219/00529B01J 2219/00527B01J 2219/00608B01J 2219/00436C40B 40/10B01J 2219/00648B01J 19/0046C07K 17/14G03F 7/38B01J 2219/00689G03F 7/265C07K 1/047B01J 2219/0059C40B 40/06C12Q 1/6874B01J 2219/00644B01J 2219/00434B01J 2219/00659B01J 2219/00531B01J 2219/00722C07K 1/042G11C 13/0019G01N 21/6458G03F 7/00C07D 317/62C07K 17/06C07H 21/00G11C 13/0014B01J 2219/00432B01J 2219/00612B01J 2219/00585B01J 2219/00637G01N 21/6452G01N 21/6428C12Q 1/6827C07H 19/04G01N 15/1433

PatentIndex Score

266

Cited by

488

References

131

Claims

Abstract

The present invention provides methods and apparatus for sequencing, fingerprinting and mapping biological macromolecules, typically biological polymers. The methods make use of a plurality of sequence specific recognition reagents which can also be used for classification of biological samples, and to characterize their sources.

Claims

exact text as granted — not AI-modified

What is claimed is:

1. A method of analyzing a receptor bound to an array of nucleic acids, comprising:
providing an array of different nucleic acids immobilized on a substrate, the different nucleic acids occupying different localized areas on the substrate of less than 0.01 cm 2 , at least some of the nucleic acids being bound to a fluorescently labeled receptor; and
detecting the localized areas of the array with a detector to detect light fluoresced from the labeled receptor bound to the nucleic acids in the localized areas, the detection step being one second or less, to detect which nucleic acids bind to the labeled receptor.

2. The method of claim 1 , wherein the detector is a charge coupled device.

3. The method of claim 1 , further comprising exposing the array to ultraviolet light.

4. The method of claim 1 , wherein each location has an area of less than 10,000 square microns.

5. The method of claim 1 , wherein each location has an area of less than 100 square microns.

6. The method of claim 1 , wherein the array comprises at least 1000 probes.

7. The method of claim 1 , wherein the combined area occupied by the at least 1000 nucleic acids is less than 1 cm 2 .

8. The method of claim 1 , wherein the array comprises at least 10 6 nucleic acids per cm 2 .

9. The method of claim 1 , wherein the nucleic acids have lengths of 2-100 nucleotides.

10. The method of claim 1 , wherein the labeled receptor is a labeled nucleic acid.

11. The method of claim 1 , wherein the labeled receptor is a labeled polypeptide.

12. The method of claim 1 , wherein the known locations are squares or rectangles.

13. The method of claim 1 , wherein the method further comprises:
directing light from a light source at the surface of the substrate; and storing a pattern of fluoresced light from the different localized areas in a data storage system.

14. The method of claim 1 , wherein the method further comprises translating the detector relative to the substrate.

15. The method of claim 1 , further comprising analyzing the stored pattern of fluoresced light to determine which nucleic acids bind to the receptor.

16. The method of claim 1 , wherein the array comprises more than 10 different nucleic acids.

17. The method of claim 1 , wherein the array comprises more than 100 different nucleic acids.

18. The method of claim 1 , wherein the array comprises more than 10,000 different nucleic acids.

19. The method of claim 1 , wherein the array comprises more than 100,000 different nucleic acids.

20. The method of claim 1 , wherein the array comprises more than 1000 different nucleic acids at a density of more than 1000 nucleic acids per cm 2 .

21. The method of claim 1 , wherein the array comprises more than 10,000 different nucleic acids at a density of more than 10,000 nucleic acids per cm 2 .

22. The method of claim 1 , wherein the array comprises more than 1,000,000 different nucleic acids at a density of more than 1,000,000 nucleic acids per cm 2 .

23. The method of claim 1 , further comprising:
illuminating the array with a device comprising:
a point light source to generate an excitation light;
an objective lens to focus the point light source at the surface of the substrate, whereby the fluorescently marked localized areas emit a fluoresced light in response to the excitation light;
a dichroic mirror to reflect light having a wavelength of the excitation light and passing light having a wavelength of the fluoresced light;
a photomultiplier and photon counter to detect the fluoresced light;
moving the substrate relative to the excitation light with an x-y translation stage; and
recording the fluoresced light as a function of a position on the surface of the substrate from which the fluoresced light was emitted with an appropriately programmed computer.

24. The method of claim 1 , further comprising washing the surface of the substrate to remove unbound labeled receptor.

25. An apparatus for analyzing ligand-receptor binding, comprising:
a substrate that comprises more than 1000 different ligands of known sequence collectively occupying less than 1 cm 2 , the different ligands occupying different localized areas within the 1 cm 2 ; and
a detector capable of detecting light from fluorescently marked localized areas on the substrate.

26. The apparatus of claim 25 , wherein the ligands are nucleic acids.

27. The apparatus of claim 25 , wherein the ligands are polypeptides.

28. The apparatus of claim 25 , wherein the ligands have known sequences.

29. An apparatus for analyzing ligand-receptor binding, comprising:
a substrate that comprises at least 100 different ligands, collectively occupying less than 1 cm 2 , the different ligands occupying different localized areas; and
a detector to detect a label bound to said ligands at said localized areas.

30. The apparatus of claim 29 , wherein the different ligands are nucleic acids.

31. The apparatus of claim 29 , wherein the different ligands have known sequences.

32. The apparatus of claim 29 , wherein the different ligands are peptides.

33. The apparatus of claim 29 , wherein the marker is a fluorescent marker.

34. The apparatus of claim 29 , wherein the marker is a radiolabelled marker.

35. An apparatus for detecting binding of nucleic acids; comprising:
(a) a substrate having a surface comprising more than 10 different nucleic acids, having known sequences, at localized areas on the surface of the substrate, each of the localized areas having an area of 10 −2 cm 2 or less, at least one of the nucleic acids being bound to a labeled receptor;
(b) an excitation light source;
(c) a detector capable of receiving a signal from fluorescently marked localized areas on the substrate;
(d) a translator to move the substrate relative to the detector; and
(e) a data collection system adapted to receive input from the detector.

36. The apparatus of claim 35 , wherein the nucleic acids have known sequences.

37. A method of analyzing a receptor bound to an array of polymers, comprising:
providing an array of different polymers immobilized on a substrate, the different polymers occupying different localized areas on the substrate having an area of less than 0.01 cm 2 , at least some of the nucleic acids being bound to a labeled receptor; and
detecting the localized areas of the array with a detector, each localized area being scanned for one second or less, to detect which polymers bind to the labeled receptor.

38. The method of claim 37 , wherein the different polymers are different nucleic acids.

39. The method of claim 37 , wherein the receptor is a nucleic acid.

40. The method of claim 37 , wherein the different polymers are peptides.

41. A method for screening comprising:
exposing a substrate having a ligand in a localized area to a marked receptor; and
detecting binding between the ligand and the marked receptor by scanning the substrate for 1 second or less in the localized area.

42. The method of claim 41 , wherein the ligand is a nucleic acid.

43. The method of claim 41 , wherein the marked receptor is a marked nucleic acid.

44. The method of claim 41 , wherein the ligand is a polypeptide.

45. The method of claim 41 , wherein the receptor is marked with a fluorescent label.

46. The method of claim 41 , wherein the substrate has more than 1000 different ligands in 1000 different localized areas within an area less than 1 cm 2 .

47. The method of claim 41 , wherein the receptor is marked with a fluorescent marker.

48. The apparatus of claim 35 , wherein the ten different nucleic acids have lengths up to 100 nucleotides.

49. An apparatus for detection of fluorescent marked locations on a substrate, wherein the substrate comprises a plurality of different polymer sequences coupled to a surface of the substrate and wherein the plurality of different polymer sequences comprises a plurality of different nucleic acid sequences, each of the different polymer sequences being coupled in a different localized area of the surface at a density of more than 1000/cm 2 , each of the localized areas having an area of 10 −3 cm 2 or less, the apparatus comprising:
(a) a light source constructed and arranged to direct light at a plurality of the localized areas;
(b) a detector constructed and arranged to detect light fluoresced from the plurality of the localized areas in response to the light source;
(c) a translator constructed and arranged to translate the substrate relative to the light source whereby the plurality of the localized areas are exposed to the light; and
(d) a data storage system constructed and arranged to store fluoresced light intensity as a function of location on the surface, the data storage system coupled to the translator and the detector.

50. An apparatus for detection of fluorescent markers at a plurality of localized areas on a substrate, comprising:
(a) a light source constructed and arranged to direct light at the plurality of localized areas;
(b) a detector constructed and arranged to detect light fluoresced from the plurality of localized areas in response to the light source;
(c) a translator constructed and arranged to translate the substrate relative to the light source whereby the plurality of localized areas are exposed to the light; and
(d) a data storage system constructed and arranged to store fluoresced light intensity as a function of location on the surface, the data storage system coupled to the translator and the detector:
wherein the substrate comprises a plurality of different polymer sequences coupled to a surface of the substrate and wherein the plurality of different polymer sequences comprises a plurality of different nucleic acid sequences, each of the different polymer sequences being coupled in a different of the localized areas, each localized area having an area of 10 −2 cm 2 or less.

51. An apparatus for detection of fluorescent markers at a plurality of localized areas on a substrate, comprising:
(a) a light source constructed and arranged to direct light at the plurality of localized areas, wherein the substrate comprises a plurality of different polymer sequences coupled to a surface of the substrate and wherein the plurality of different polymer sequences comprises a plurality of different nucleic acid sequences, each of the different polymer sequences being coupled in a different of the localized areas, each of the localized areas having an area of 10 −2 cm 2 or less;
(b) a detector constructed and arranged to detect light fluoresced from the plurality of localized areas in response to the light source;
(c) a translator constructed and arranged to translate the substrate relative to the light source whereby the plurality of localized areas are exposed to the light; and
(d) a data storage system constructed and arranged to store fluoresced light intensity as a function of location on the surface, the data storage system coupled to the translator and the detector.

52. A method in accordance with claim 1 further comprising collecting and analyzing data from the detection step using a computer system.

53. A method in accordance with claim 52 wherein the computer system comprises an IBM PC or AT compatible computer.

54. A method in accordance with claim 1 further comprising translating the array using an X/Y translation table.

55. A method in accordance with claim 54 wherein the X/Y translation table is controlled by a digital computer.

56. A method in accordance with claim 1 wherein the detecting step uses a photomultiplier tube.

57. A method in accordance with claim 1 wherein the detecting step uses a pre-amplifier.

58. A method in accordance with claim 1 further comprising displaying data that has been acquired using a video display.

59. A method in accordance with claim 1 further comprising detecting and displaying the output of the detection using a photomultiplier tube, a pre-amplifier, a computer, and a video display.

60. A method in accordance with claim 1 wherein the detecting step involves counting the number of photons per unit area.

61. A method in accordance with claim 1 wherein the detecting step includes acquiring data every 1 to 100 μm.

62. A method in accordance with claim 1 wherein the detection step includes acquiring data with a data collection diameter of about 0.8 to 10 μm.

63. A method in accordance with claim 1 further comprising exposing the array to laser light.

64. A method in accordance with claim 1 further comprising exposing the array to laser light having a wavelength of greater than 520 nm.

65. A method in accordance with claim 1 further comprising translating the array with an X/Y translator under the control of a computer; detecting and displaying the output of the detection using a photomultiplier tube, a pre-amplifier, and a computer; and collecting and analyzing data from the detection step using a computer system.

66. A method in accordance with claim 37 further comprising collecting and analyzing data from the detection step using a computer system.

67. A method in accordance with claim 66 wherein the computer system comprises an IBM PC or AT compatible computer.

68. A method in accordance with claim 37 further comprising translating the array using an X/Y translation table.

69. A method in accordance with claim 37 wherein the X/Y translation table is controlled by a digital computer.

70. A method in accordance with claim 37 wherein the detecting step uses a photomultiplier tube.

71. A method in accordance with claim 37 wherein the detecting step uses a pre-amplifier.

72. A method in accordance with claim 37 further comprising displaying data that has been acquired using a video display.

73. A method in accordance with claim 37 further comprising detecting and displaying the output of the detection using a photomultiplier tube, a pre-amplifier, a computer, and a video display.

74. A method in accordance with claim 37 wherein the detecting step involves counting the number of photons per unit area.

75. A method in accordance with claim 37 wherein the detecting step includes acquiring data every 1 to 100 μm.

76. A method in accordance with claim 37 wherein the detection step includes acquiring data with a data collection diameter of about 0.8 to 10 μm.

77. A method in accordance with claim 37 further comprising exposing the array to laser light.

78. A method in accordance with claim 37 further comprising exposing the array to laser light having a wavelength of greater than 520 nm.

79. A method in accordance with claim 37 further comprising translating the array with an X/Y translator under the control of a computer; detecting and displaying the output of the detection using a photomultiplier tube, a pre-amplifier, and a computer; and collecting and analyzing data from the detection step using a computer system.

80. An apparatus in accordance with claim 37 further comprising a computer system.

81. An apparatus in accordance with claim 80 wherein the computer system comprises an IBM PC or AT compatible computer.

82. An apparatus in accordance with claim 25 further comprising an X/Y translation table.

83. An apparatus in accordance with claim 25 wherein the X/Y translation table is controlled by a digital computer.

84. An apparatus in accordance with claim 25 further comprising a photomultiplier tube.

85. An apparatus in accordance with claim 25 further comprising a pre-amplifier.

86. An apparatus in accordance with claim 25 further comprising a video display.

87. An apparatus in accordance with claim 25 further comprising a photomultiplier tube, a pre-amplifier, a computer, and a video display.

88. An apparatus in accordance with claim 29 further comprising a laser.

89. An apparatus in accordance with claim 29 further comprising an X/Y translator under the control of a computer; a photomultiplier tube, a pre-amplifier, and a computer; and a computer system.

90. An apparatus in accordance with claim 29 further comprising a computer system.

91. An apparatus in accordance with claim 90 wherein the computer system comprises an IBM PC or AT compatible computer.

92. An apparatus in accordance with claim 37 further comprising an X/Y translation table.

93. An apparatus in accordance with claim 92 wherein the X/Y translation table is controlled by a digital computer.

94. An apparatus in accordance with claim 29 further comprising a photomultiplier tube.

95. An apparatus in accordance with claim 29 further comprising a pre-amplifier.

96. An apparatus in accordance with claim 29 further comprising a video display.

97. An apparatus in accordance with claim 29 further comprising a photomultiplier tube, a pre-amplifier, a computer, and a video display.

98. An apparatus in accordance with claim 29 further comprising a laser.

99. An apparatus in accordance with claim 29 further comprising an X/Y translator under the control of a computer; a photomultiplier tube, a pre-amplifier, and a computer; and a computer system.

100. A method in accordance with claim 23 wherein the receptor is a nucleic acid.

101. An apparatus in accordance with claim 25 wherein the receptor is a nucleic acid.

102. An apparatus in accordance with claim 29 wherein the receptor is a nucleic acid.

103. A method in accordance with claim 25 wherein the receptor is a peptide.

104. A method in accordance with claim 37 wherein the receptor is a peptide.

105. A method in accordance with claim 29 wherein the receptor is a peptide.

106. A method for detecting binding between a nucleic acid probe bound to a substrate and a nucleic acid target comprising:
providing an array of positionally distinguishable nucleic acids of known sequence bound to a substrate having a density of more than 300 different nucleic acids/cm 2 ; and
detecting a double stranded nucleic acid.

107. A method in accordance with claim 106 wherein the density of the array is 1,000 different nucleic acids/cm 2 .

108. A method in accordance with claim 107 wherein the density of the array is more than 10,000 different nucleic acids/cm 2 .

109. A method of analyzing using an array of nucleic acids, comprising:
providing an array of different nucleic acids of known sequence immobilized on a substrate at a density more than 1,000 nucleic acids/cm 2 , the different nucleic acids being positionally distinguishable and, at least some of the nucleic acids being bound to a fluorescently labeled receptor;
illuminating an area of the array greater than 10 −3 cm 2 with an energy source; and
detecting fluoresced light from the known locations of the array with a detector, the detection step being one second or less, to detect which nucleic acids bind to the labeled receptor.

110. A method in accordance with claim 109 wherein the receptor is a nucleic acid.

111. A method in accordance with claim 109 wherein the receptor is a peptide.

112. A method in accordance with claim 109 wherein the density is more than 10,000 nucleic acids/cm 2 .

113. A method in accordance with claim 1 wherein the sequences of the nucleic acids are known.

114. An apparatus in accordance with claim 41 wherein the sequence of the ligand is known.

115. An apparatus in accordance with claim 49 wherein the sequences of the nucleic acids are known.

116. An apparatus in accordance with claim 50 wherein the sequences of the nucleic acids are known.

117. An apparatus in accordance with claim 51 wherein the sequences of the nucleic acids are known.

118. An apparatus in accordance with claim 35 wherein the density of the different nucleic acid sequences on the array is more than 1000/cm 2 .

119. An apparatus in accordance with claim 37 wherein the density of the different nucleic acid sequences on the array is more than 1000/cm 2 .

120. An apparatus in accordance with claim 41 wherein the density of the different nucleic acid sequences on the array is more than 1000/cm 2 .

121. An apparatus in accordance with claim 50 wherein the density of the different nucleic acid sequences on the array is more than 1000/cm 2 .

122. An apparatus in accordance with claim 51 wherein the density of the different nucleic acid sequences on the array is more than 1000/cm 2 .

123. An apparatus in accordance with claim 1 wherein the density of the different nucleic acid sequences on the array is more than 300/cm 2 .

124. An apparatus in accordance with claim 1 wherein the density of the different nucleic acid sequences on the array is more than 300/cm 2 .

125. An apparatus in accordance with claim 35 wherein the density of the different nucleic acid sequences on the array is more than 300/cm 2 .

126. An apparatus in accordance with claim 29 wherein the density of the different nucleic acid sequences on the array is more than 300/cm 2 .

127. A method of analyzing using an array of polymers, comprising:
providing an array of different polymers of known sequence immobilized on a substrate at a density more than 1,000 polymers/cm 2 , the different polymers being positionally distinguishable and at least some of the polymers being bound to a fluorescently labeled target polymers;
illuminating an area of the array greater than 10 −3 cm 2 with an energy source; and
detecting fluoresced light from the known locations of the array with a detector, the detection step being one second or less, to detect which polymers on the array bind to the labeled target polymers.

128. A method in accordance with claim 127 wherein the polymers are nucleic acids.

129. A method in accordance with claim 127 wherein the polymers are peptides.

130. A method in accordance with claim 128 the density of the different nucleic acids is more than 10,000/cm 2 .

131. A method in accordance with claim 129 wherein the density of the different peptides is more than 10,000/cm 2 .

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